Age and sex effects across the blood proteome after ionizing radiation exposure can bias biomarker screening and risk assessment

Molecular biomarkers of ionizing radiation (IR) exposure are a promising new tool in various disciplines: they can give necessary information for adaptive treatment planning in cancer radiotherapy, enable risk projection for radiation-induced survivorship diseases, or facilitate triage and intervention in radiation hazard events. However, radiation biomarker discovery has not yet resolved the most basic features of personalized medicine: age and sex. To overcome this critical bias in biomarker identification, we quantitated age and sex effects and assessed their relevance in the radiation response across the blood proteome. We used high-throughput mass spectrometry on blood plasma collected 24 h after 0.5 Gy total body irradiation (15 MV nominal photon energy) from male and female C57BL/6 N mice at juvenile (7-weeks-old) or adult (18-weeks-old) age. We also assessed sex and strain effects using juvenile male and female BALB/c nude mice. We showed that age and sex created significant effects in the proteomic response regarding both extent and functional quality of IR-induced responses. Furthermore, we found that age and sex effects appeared non-linear and were often end-point specific. Overall, age contributed more to differences in the proteomic response than sex, most notably in immune responses, oxidative stress, and apoptotic cell death. Interestingly, sex effects were pronounced for DNA damage and repair pathways and associated cellular outcome (pro-survival vs. pro-apoptotic). Only one protein (AHSP) was identified as a potential general biomarker candidate across age and sex, while GMNN, REG3B, and SNCA indicated some response similarity across age. This low yield advocated that unisex or uniage biomarker screening approaches are not feasible. In conclusion, age- and sex-specific screening approaches should be implemented as standard protocol to ensure robustness and diagnostic power of biomarker candidates. Bias-free molecular biomarkers are a necessary progression towards personalized medicine and integral for advanced adaptive cancer radiotherapy and risk assessment.

p. 6-8 Figure S2 p. 9 Table S1 p. 10 Table S2 p. 11 Table S3 p. 12 Table S4 p. 13 Table S5 p. 14 Table S6 p. 15 Table S7 p. 16 Table S8 p. 17 washed 2 times with 200 µl of 8 M urea. Alkylation of the reduced cysteine side chains was performed with 10 mM methyl methanethiosulfonate (MMTS) diluted in digestion buffer (1% sodium deoxycholate (SDC), 50 mM TEAB) for 30 min at room temperature and the filters were then repeatedly washed with digestion buffer. Trypsin in digestion buffer was added (500 ng for analysis 1 or 800 ng for analysis 2) and the sample was incubated at 37 °C overnight, then another 500/800 ng portion of trypsin was added followed by the incubation for 2 h (analysis 1) or 3 h (analysis 2). Digested peptides were collected at 10,000 rpm for 20 min, followed by a wash with 20 µl of the digestion buffer and centrifugation at 10,000 rpm for 20 min. In short, the immunodepleted plasma samples were reduced with DLdithiothreitol, alkylated with methyl methanethiosulfonate, digested with trypsin in presence of 1% sodium deoxycholate. The resulting peptides were labelled using the TMT 10plex isobaric reagents according to the manufacturer's instructions (Thermo Scientific), combined into one TMT set, concentrated using vacuum centrifugation and SDC was removed by acidification with 10% trifluoroacetic acid (TFA) and subsequent centrifugation at 13,000 rpm for 10 min.

Liquid Chromatography-Mass Spectrometry Analysis
All samples were analyzed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific Precursor MS scans were performed at 120,000 resolution; the most abundant precursors with charges 2 to 7 were selected for fragmentation over the 3 s cycle time, fragmented by collision induced dissociation (CID) at 30% (analysis 1) or 35% (analysis 2) collision energy, and the MS 2 spectra were detected in the ion trap followed by the synchronous isolation of the 5 most abundant MS 2 fragment ions and fragmentation by higher-energy collision dissociation (HCD); the resulting MS 3 spectra were detected in the Orbitrap at 60,000 (analysis 1) or 50,000 (analysis 2) resolution.

Data analysis Data Availability
The detailed proteomic sample preparation protocol and the LC-MS analysis parameters are described in the Electronic Supplementary Information. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015859 (2).

Proteomic data analysis
Peptide and protein identification and quantification were performed using Proteome    Table S8.

Group Functional category (number of proteins)
Differentially abundant proteins with fold change (FC) values |FC| ≥ 3 were subjected to enrichment analysis using Gene Ontology terms (http://geneontology.org/). Based on GO annotations, proteins were grouped into functional categories representing hallmarks of ionizing radiation-induced responses. The number of proteins associated with respective cellular functions is shown for each group.   Differentially abundant proteins with fold change (FC) values |FC| ≥ 3 were subjected to enrichment analysis using Gene Ontology (GO) terms (http://geneontology.org/). Based on GO annotations, proteins were grouped into functional categories representing hallmarks of ionizing radiation-induced responses.   Differentially abundant proteins with fold change (FC) values |FC| ≥ 3 were subjected to enrichment analysis using Gene Ontology (GO) terms (http://geneontology.org/). Based on GO annotations, proteins were grouped into functional categories representing hallmarks of ionizing radiation-induced responses. Differentially abundant proteins with fold change (FC) values |FC| ≥ 3 were subjected to enrichment analysis using Gene Ontology (GO) terms (http://geneontology.org/). Based on GO annotations, proteins were grouped into functional categories representing hallmarks of ionizing radiation-induced responses.